We are proposing studies to determine the resolution limits for fluorescence diffuse optical tomography (FDOT) using over-sampled measurements. It will exploit the combined expertise of two NCRR National Resources: data acquisition using the recently established Molecular Imaging Workbench at the Duke Center for In Vivo Microscopy (CIVM) and image reconstruction using the parallel computing facilities at the Pittsburgh Supercomputer Center (PSC). Specifically, we will be conducting the following studies: (i) Develop a modular open-source software package for FDOT simulations and image reconstruction;(ii) Determine FDOT resolution limits in imaging phantoms;and (iii) Determine the resolution limits with prior anatomical information and apply the technology to lung cancer imaging in mice. PUBLIC HEALTH RELEVANCE (provided by applicant): The goal of this proposal is to investigate the resolution limit in 3D fluorescence imaging for small animals. The effects of anatomical prior information from micro-CT will also be studied. Image reconstruction will be based on a Monte Carlo method using parallel computing on supercomputers.